**5. Genotyping of VNTR DRD4 gene polymorphism**

Genotyping was performed according to a modified technique of Nanko et al. [23]. A DNA fragment of the DRD4 gene (GenBank identification number L12398) containing a DNA region with a variable size of 96–384 bp was amplified using primers: 5′-AGGTG-GCACG-TCGCG-CCAAG-CTGCA-3 'straight, pos. 668–692; 5'-TCTGC-GGTGG-AGTCT-GGGGT-GGGAG-3 'reverse, pos. 1129–1105. The amplification reaction mixture contained 0.5–1 μg of genomic DNA, direct and reverse primers at a concentration of 0.4 μM each, dNTP at a concentration of 0.1 mm each, 1.5 mm MgCl2, 10% dimethyl sulfoxide (DMSO), 0.01% by volume Tween-20, 20 mm (NH4) SO4, 75 mM TrisHCl (pH 9.0) and 1.25 units of Taq polymerase. The total volume of the mixture was 25 μl. PCR was performed using a Mastercycler gradient (Eppendorf Scientific Inc., USA). The amplification conditions were as follows: 95°C for 4 min, then 35 cycles: 95°C for 1 min, 65°C for 1 min, 72°C for 1 min. PCR products were analyzed using polyacrylamide gel electrophoresis (4%), in a buffer containing 90 mM Tris-borate (pH 8.0) and 2 mM EDTA, followed by staining with ethidium bromide. DNA markers of 100 bp were used as a molecular weight marker. (Sibenzyme, Russia).

## **6. Genotyping of VNTR DAT gene polymorphism**

For genotyping, we used a modified method of Mitchell et al. [24]. The DNA fragment of the DAT gene (identification number in GenBank M95167), containing a DNA region with a variable size of 240–480 bp, was amplified using primers: 5′-TGTGG-TGTAG- GGAAC-GGCCT-GAG-3 'straight, pos. 2718–2740; 5'-CTTCC-TGGAG-GTCAC-GGCTC-AAGG-3 'reverse, pos. 3201–3178. The reaction mixture

#### *Association of Personal Anxiety with Dopamine Receptor D4 (DRD4), DAT Genes Polymorphism DOI: http://dx.doi.org/10.5772/intechopen.94386*

with a volume of 25 μl contained 0.5–1 μg of genomic DNA, forward and reverse primers at a concentration of 0.4 μM each, dNTP at a concentration of 0.4 mm each, 2 mm MgCl2, 0.01% by volume Tween-20, 98 mm beta mercaptoethanol, 67 mm Tris HCl (pH 8.8) and 1.25 units of Taq polymerase. Each of the 35 amplification cycles consisted of denaturation (95°C, 0.5 min), annealing (66°C, 5 min) and synthesis (72°C, 1.5 min). PCR products were analyzed using polyacrylamide gel electrophoresis (4%), followed by staining with ethidium bromide. DNA markers of 100 bp were used as a molecular weight marker. (Sibenzyme, Russia).

Statistical analysis was performed using the SPSS-19 software package [25]. The distribution of attributes and their numerical characteristics were analyzed. The analysis of simple relationships between variables (contingency tables) was carried out. Using the method of constructing contingency tables, we conducted the hypothesis of factors A and B independence or the homogeneity of factor B with respect to the levels of factor A. The reliability of the factor independence was evaluated using χ<sup>2</sup> criterion [26].
